Extracellular vesicle-aso constructs targeting stat6

Abstract

The present disclosure relates to extracellular vesicles, e.g., exosomes, comprising an antisense oligonucleotide (ASO), wherein the ASO comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within a STAT6 transcript. Also provided herein are methods for producing the exosomes and methods for using the exosomes to treat and/or prevent diseases or disorders.

What is claimed: 
     
         1 . A method of preventing or treating a disease or condition in a subject in need thereof, comprising administering to the subject (i) an extracellular vesicle comprising an antisense oligonucleotide (ASO) and (ii) a programmed death-1 (PD-1) antagonist; wherein the ASO comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within a target transcript, wherein the target transcript is a STAT6 transcript (SEQ ID NO: 1 or SEQ ID NO: 3). 
     
     
         2 . A method of increasing or enhancing an immune response in a subject in need thereof, comprising administering to the subject (i) an extracellular vesicle comprising an antisense oligonucleotide (ASO) and (ii) a programmed death-1 (PD-1) antagonist; wherein the ASO comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is complementary to a nucleic acid sequence within a target transcript, wherein the target transcript is a STAT6 transcript (SEQ ID NO: 1 or SEQ ID NO: 3). 
     
     
         3 . The method of  claim 1  or  2 , wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within nucleotides 1 to 2056 of a STAT6 transcript corresponding to a nucleotide sequence as set forth in SEQ ID NO: 3 or nucleotides 2059 to 3963 of a STAT6 transcript corresponding to a nucleotide sequence as set forth in SEQ ID NO: 3. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the ASO is a gapmer, a mixmer, or a totalmer. 
     
     
         5 . The method of any one of  claims 1  to  4 , wherein the ASO comprises one or more nucleoside analogs. 
     
     
         6 . The method of  claim 5 , wherein one or more of the nucleoside analogs comprises a 2′-O-alkyl-RNA; 2′-O-methyl RNA (2′-OMe); 2′-alkoxy-RNA; 2′-O-methoxyethyl-RNA (2′-MOE); 2′-amino-DNA; 2′-fluro-RNA; 2′-fluoro-DNA; arabino nucleic acid (ANA); 2′-fluoro-ANA; or bicyclic nucleoside analog. 
     
     
         7 . The method of  claim 5  or  6 , wherein one or more of the nucleoside analogs is a sugar modified nucleoside. 
     
     
         8 . The method of  claim 7 , wherein the sugar modified nucleoside is an affinity enhancing 2′ sugar modified nucleoside. 
     
     
         9 . The method of any one of  claims 5  to  8 , wherein one or more of the nucleoside analogs comprises a nucleoside comprising a bicyclic sugar. 
     
     
         10 . The method of any one of  claims 5  to  8 , wherein one or more of the nucleoside analogs comprises an LNA. 
     
     
         11 . The method of any one of  claims 5  to  10 , wherein one or more of the nucleotide analogs is selected from the group consisting of constrained ethyl nucleoside (cEt), 2′,4′-constrained 2′-O-methoxyethyl (cMOE), α-L-LNA, β-D-LNA, 2′-O,4′-C-ethylene-bridged nucleic acids (ENA), amino-LNA, oxy-LNA, thio-LNA, and any combination thereof. 
     
     
         12 . The method of any one of  claims 1  to  11 , wherein the ASO comprises one or more 5′-methyl-cytosine nucleobases. 
     
     
         13 . The method of any one of  claims 1  to  12 , wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within (i) a 5′ untranslated region (UTR); (ii) a coding region; or (iii) a 3′ UTR of the target transcript. 
     
     
         14 . The method of any one of  claims 1  to  13 , wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence comprising (i) nucleotides 1-700 of SEQ ID NO: 3; (ii) nucleotides 1000-1500 of SEQ ID NO: 3; (iii) nucleotides 1500-2000 of SEQ ID NO: 3; (iv) nucleotides 2000-2500 of SEQ ID NO: 3; (v) 2500-3000 of SEQ ID NO: 3; (vi) 3000-3700 of SEQ ID NO: 3, (vii) nucleotides 413-803 of SEQ ID NO: 3; (viii) nucleotides 952-1688 of SEQ ID NO: 3; (ix) nucleotides 1726-2489 of SEQ ID NO: 3; (x) nucleotides 2682-2912 of SEQ ID NO: 3; (xi) 2970-3203 of SEQ ID NO: 3; (xii) 3331-3561 of SEQ ID NO: 3; (xiii) nucleotides 463-753 of SEQ ID NO: 3; (xiv) nucleotides 1002-1638 of SEQ ID NO: 3; (xv) nucleotides 1776-2439 of SEQ ID NO: 3; (xvi) nucleotides 2682-2862 of SEQ ID NO: 3; (xvii) 3020-3153 of SEQ ID NO: 3; (xviii) 3381-3511 of SEQ ID NO: 3; (xix) nucleotides 503-713 of SEQ ID NO: 3; (xx) nucleotides 1042-1598 of SEQ ID NO: 3; (xxi) nucleotides 1816-2399 of SEQ ID NO: 3; (xxii) nucleotides 2722-2822 of SEQ ID NO: 3; (xxiii) 3060-3113 of SEQ ID NO: 3; or (xxiv) 3421-3471 of SEQ ID NO: 3. 
     
     
         15 . The method of any one of  claims 1  to  14 , wherein the contiguous nucleotide sequence is complementary to a nucleic acid sequence within (i) nucleotides 513-703 of SEQ ID NO: 3; (ii) nucleotides 1052-1588 of SEQ ID NO: 3; (iii) nucleotides 1826-2389 of SEQ ID NO: 3; (iv) nucleotides 2732-2812 of SEQ ID NO: 3; (v) 3070-3103 of SEQ ID NO: 3; or (vi) 3431-3461 of SEQ ID NO: 3. 
     
     
         16 . The method of any one of  claims 1  to  15 , wherein the ASO comprises a nucleic acid sequence selected from GAAAGGTTCCGTCGGGC (SEQ ID NO: 144), CTGAGTCGCTGAAGCGG (SEQ ID NO: 145), GCCCTTGTACTTTTGCATAG (SEQ ID NO: 193), GCAAGATCCCGGATTCGGTC (SEQ ID NO: 185), and any combination thereof. 
     
     
         17 . The method of any one of  claims 1  to  16 , wherein the contiguous nucleotide sequence comprises a nucleotide sequence complementary to a sequence selected from the sequences in  FIG.  1   . 
     
     
         18 . The method of any one of  claims 1  to  17 , wherein the continuous nucleotide sequence is fully complementary to a nucleotide sequence within the target transcript. 
     
     
         19 . The method of any one of  claims 1  to  18 , wherein the ASO comprises a nucleotide sequence selected from SEQ ID NOs: 91-193, with one or two mismatches. 
     
     
         20 . The method of any one of  claims 1  to  19 , wherein the ASO has a design selected from the group consisting of the designs in  FIG.  1   , wherein the upper letter is a sugar modified nucleoside and the lower case letter is DNA. 
     
     
         21 . The method of any one of  claims 1  to  20 , wherein the ASO is from 14 to 20 nucleotides in length. 
     
     
         22 . The method of any one of  claims 1  to  21 , wherein the contiguous nucleotide sequence comprises one or more modified internucleoside linkages. 
     
     
         23 . The method of  claim 22 , wherein the one or more modified internucleoside linkages is a phosphorothioate linkage. 
     
     
         24 . The method of  claim 22  or  23 , wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of internucleoside linkages are modified. 
     
     
         25 . The method of  claim 24 , wherein each of the internucleoside linkages in the ASO is a phosphorothioate linkage. 
     
     
         26 . The method of any one of  claims 1  to  25 , wherein the ASO is linked to an anchoring moiety. 
     
     
         27 . The method of  claim 26 , wherein the anchoring moiety comprises a sterol, GM1, a lipid, a vitamin, a small molecule, a peptide, or a combination thereof. 
     
     
         28 . The method of  claim 26  or  27 , wherein the anchoring moiety comprises cholesterol. 
     
     
         29 . The method of any one of  claims 25  to  27 , wherein the anchoring moiety comprises a phospholipid, a lysophospholipid, a fatty acid, a vitamin (e.g., vitamin D and/or vitamin E), or any combination thereof. 
     
     
         30 . The method of any one of  claims 26  to  29 , wherein the anchoring moiety is associated with the extracellular vesicle. 
     
     
         31 . The method of any one of  claims 26  to  30 , wherein the extracellular vesicle comprises a lipid bilayer, wherein the anchoring moiety is associated with the lipid bilayer of the extracellular vesicle. 
     
     
         32 . The method of any one of  claims 26  to  31 , wherein the ASO is linked to the anchoring moiety on the exterior surface of the EV. 
     
     
         33 . The method of any one of  claims 26  to  31 , wherein the ASO is linked to the anchoring moiety on the luminal surface of the EV. 
     
     
         34 . The method of any one of  claims 26  to  31 , wherein the ASO is linked to the anchoring moiety. 
     
     
         35 . The method of any one of  claims 26  to  31 , wherein the anchoring moiety comprises a scaffold moiety. 
     
     
         36 . The method of any one of  claims 1  to  35 , wherein the ASO is linked to the EV by a linker. 
     
     
         37 . The method of  claim 35  or  36 , wherein the linker is a polypeptide. 
     
     
         38 . The method of  claim 35  or  36 , wherein the linker is a non-polypeptide moiety. 
     
     
         39 . The method of  claim 35  or  36 , wherein the linker comprise ethylene glycol. 
     
     
         40 . The method of  claim 39 , wherein the linker comprises HEG, TEG, PEG, or any combination thereof. 
     
     
         41 . The method of  claim 35  or  36 , wherein the linker comprises acrylic phosphoramidite (e.g., ACRYDITE™), adenylation, azide (NHS Ester), digoxigenin (NHS Ester), cholesterol-TEG, I-LINKER™, an amino modifier (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, or Uni-Link™ amino modifier), alkyne, 5′ Hexynyl, 5-Octadiynyl dU, biotinylation (e.g., biotin, biotin (Azide), biotin dT, biotin-TEG, dual biotin, PC biotin, or desthiobiotin), thiol modification (thiol modifier C3 S—S, dithiol or thiol modifier C6 S—S), or any combination thereof. 
     
     
         42 . The method of any one of  claims 35  to  41 , wherein the linker is a cleavable linker. 
     
     
         43 . The method of  claim 42 , wherein the linker comprises valine-alanine-p-aminobenzylcarbamate or valine-citrulline-p-aminobenzylcarbamate. 
     
     
         44 . The method of any one of  claims 35  to  43 , wherein the linker comprises (i) a maleimide moiety and (ii) valine-alanine-p-aminobenzylcarbamate or valine-citrulline-p-aminobenzylcarbamate. 
     
     
         45 . The method of any one of  claims 1  to  44 , wherein the extracellular vesicle further comprises an exogenous targeting moiety. 
     
     
         46 . The method of  claim 45 , wherein the exogenous targeting moiety comprises a peptide, an antibody or an antigen-binding fragment thereof, a chemical compound, an RNA aptamer, or any combination thereof. 
     
     
         47 . The method of  claim 45  or  46 , wherein the exogenous targeting moiety comprises a peptide. 
     
     
         48 . The method of any one of  claims 45  to  37 , wherein the exogenous targeting moiety comprises a microprotein, a designed ankyrin repeat protein (darpin), an anticalin, an adnectin, an aptamer, a peptide mimetic molecule, a natural ligand for a receptor, a camelid nanobody, or any combination thereof. 
     
     
         49 . The method of any one of  claims 45  to  48 , wherein the exogenous targeting moiety comprises a full-length antibody, a single domain antibody, a heavy chain only antibody (VHH), a single chain antibody, a shark heavy chain only antibody (VNAR), an scFv, a Fv, a Fab, a Fab′, a F(ab′)2, or any combination thereof. 
     
     
         50 . The method of  claim 52 , wherein the antibody is a single chain antibody. 
     
     
         51 . The method of any one of  claims 45  to  50 , wherein the exogenous targeting moiety targets the exosome to the liver, heart, lungs, brain, kidneys, central nervous system, peripheral nervous system, muscle, bone, joint, skin, intestine, bladder, pancreas, lymph nodes, spleen, blood, bone marrow, or any combination thereof. 
     
     
         52 . The method of any one of  claims 45  to  51 , wherein the exogenous targeting moiety targets the extracellular vesicle to a tumor cell, dendritic cell, T cell, B cell, macrophage, neuron, hepatocyte, Kupffer cell, myeloid-lineage cell (e.g., a neutrophils, monocytes, macrophages, hematopoietic stem cell, an MDSC (e.g., a monocytic MDSC or a granulocytic MDSC)), or any combination thereof. 
     
     
         53 . The method of any one of  claims 45  to  51 , wherein the extracellular vesicle comprises a scaffold moiety linking the exogenous targeting moiety to the extracellular vesicle. 
     
     
         54 . The method of  claim 35  or  53 , wherein the scaffold moiety is a Scaffold X. 
     
     
         55 . The method of  claim 35  or  54 , wherein the scaffold moiety is a Scaffold Y. 
     
     
         56 . The method of any one of  claims 1  to  55 , wherein the extracellular vesicle is an exosome. 
     
     
         57 . The method of any one of  claims 1  to  56 , wherein the PD-1 antagonist comprises an antibody or an antigen-binding portion thereof that specifically bind to human PD-1 and blocks or inhibits the interaction between PD-1 and PD-L1 (“an anti-PD-1 antibody”). 
     
     
         58 . The method of  claim 57 , wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, PDR001, MEDI-0680, cemiplimab, JS001, BGB-A317, INCSHR1210, TSR-042, GLS-010, AM-0001, STI-1110, AGEN2034, MGA012, IBI308, and any combination thereof. 
     
     
         59 . The method of any one of  claims 1  to  56 , wherein the PD-1 antagonist comprises an antibody or an antigen-binding portion thereof that specifically bind to human PD-L1 and blocks or inhibits the interaction between PD-1 and PD-L1 (“an anti-PD-L1 antibody”). 
     
     
         60 . The method of  claim 59 , wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, STI-1014, CX-072, KN035, LY3300054, CK-301, BMS-936559, and any combination thereof. 
     
     
         61 . The method of any one of  claims 1  to  60 , wherein the extracellular vesicle and the PD-1 antagonist are administered concurrently. 
     
     
         62 . The method of any one of  claims 1  to  61 , wherein the extracellular vesicle and the PD-1 antagonist are administered sequentially. 
     
     
         63 . The method of  claim 62 , wherein the extracellular vesicle and the PD-1 antagonist are administered on different days. 
     
     
         64 . The method of any one of  claims 1  to  63 , wherein the PD-1 antagonist is linked to or associated with the extracellular vesicle. 
     
     
         65 . The method of any one of  claims 1  to  64 , wherein the extracellular vesicle, the PD-1 antagonist, or both are administered by a route selected from the groups consisting of parenteral administration, topical administration, intravenous administration, oral administration, subcutaneous administration, intra-arterial administration, intradermal administration, transdermal administration, rectal administration, intracranial administration, intraperitoneal administration, intrathecal administration, intranasal administration, intratumoral administration, intramuscular administration, inhalation, and any combination thereof. 
     
     
         66 . The method of any one of  claims 1  to  65 , wherein the subject is afflicted with a cancer. 
     
     
         67 . The method of  claim 66 , wherein the cancer is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell cancer, squamous cell cancer of the head and neck cancer, colorectal cancer, lymphoma, leukemia, liver cancer, glioblastoma, melanoma, myeloma basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary cancer, papillary adenocarcinomas, cystadenocarcinoma, medullary cancer, bronchogenic cancer, renal cell cancer, hepatoma, bile duct cancer, choriocarcinoma, seminoma, nonseminoma, embryonal cancer, Wilms' tumor, cervical cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, follicular lymphoma, Hodgkin's lymphoma, B cell lymphoma, and any combination thereof.